The European corn borer Ostrinia nubilalis (Hübner) is a model of evolution of sexual communication in insects. Two pheromone strains produce and respond to opposite ratios of the two pheromone components, Z11 and E11-tetradecenylacetate. The Z-strain uses a ratio of 97:3 of Z11:E11 tetradecenylacetate, whereas the E-strain uses a ratio of 1:99. We studied how the difference in male preference correlates with differences in wiring of olfactory input and output neurons in the antennal lobe (AL). Activity-dependent anterograde staining, intracellular recording and immunocytochemistry were used to establish the structure and function of male olfactory receptor neurons (ORNs) and AL projection neurons (PNs). Physiologically characterized neurons were reconstructed using confocal microscopy of alpha-synapsin stained ALs. The ALs of males and females in both strains had approximately 64 glomeruli. In males the macroglomerular complex (MGC) was morphologically similar in the two strains and consisted of two major compartments, a large, medial compartment folded around a smaller, lateral one. Extensive physiological and morphological analysis revealed that in both strains the major pheromone component-specific ORNs and PNs arborize in the medial MGC glomerulus, whereas those sensitive to the minor pheromone component arborize in the lateral glomerulus. In other words, the two strains have an indistinguishable MGC morphology, but a reversed topology. Apparently, the single-gene-mediated shift that causes a radical change in behavior is located upstream of the antennal lobes, i.e. at the ORN level.